Protein phosphorylation plays a critical role in the regulation of proliferation and differentiation in eukaryotes. While there is a rich literature on these regulatory pathways in higher eukaryotes, their roles in pathogenic microbes is only poorly understood....." Dr. Parsons has chosen to study Trypanosoma brucei, a protozoan parasite that causes human sleeping sickness and related diseases in animals. Dr. Parsons will employ an integrated approach to studying signal transduction in T. brucei, using molecular biology, protein chemistry, immunochemistry, and microscopy to examine two pathways: the Nrk pathway and the pp44/46 pathway. We have identified initial reference molecules and now will progress to the study of the upstream and downstream elements of the pathways. One reference molecule is the developmentally regulated Nrk protein kinase, which is under transnational control. As the immediate upstream regulatory process, this transnational control will be further studied. Other key features of Nrk are its PH domain (a modular domain involved in protein-protein interactions and membrane localization) and its apparent association with a phosphoprotein, pp52. The investigator will analyze the association of Nrk with other molecules and determine if Nrk is membrane-associated. The downstream events in the pathway will be examined through genetic modification of Nrk expression in vivo. pp44/46 is a tyrosine/serine phosphorylated protein that is located in a subnuclear compartment. Its sequence shows features of nucleolar RNA binding proteins and the protein is capable of binding nucleic acid. We will determine if pp44/46 resides in the nucleolus and characterize its interaction with nucleic acids. Although not a kinase itself, pp44/46 is associated with serine/tyrosine kinase activity. Dr. Parsons will initiate experiments aimed at cloning this kinase using the yeast two-hybrid system.